The present invention relates generally to the use of electric pulses to increase the permeability of cell, and more specifically to a method and electroporation therapy apparatus for the application of controlled electric fields for delivery of agents into cells by electroporation.
In the 1970""s it was discovered that electric fields could be used to create pores in cells without causing permanent damage. This discovery made possible the insertion of large molecules into cell cytoplasm. It is known that genes and other molecules such as pharmacological compounds can be incorporated into live cells through a process known as electroporation. The genes or other molecules are mixed with the live cells in a buffer medium and short pulses of high electric fields are applied. The cell membranes are transiently made porous and the genes or molecules enter the cells, where they can modify the genome of the cell.
Electroporation in vivo is often limited to tissue or cells that are close to the skin of the organism where the electrodes can be placed. Therefore, tissue which would otherwise be treatable by systemic drug delivery or chemotherapy, such as a tumor, is generally inaccessible to electrodes used for electroporation. In the treatment of certain types of cancer with chemotherapy, it is necessary to use a high enough dose of a drug to kill the cancer cells without killing an unacceptable high number of normal cells. If the chemotherapy drug could be inserted directly inside the cancer cells, this objective could be achieved. Some of the anti-cancer drugs, for example, bleomycin, normally cannot penetrate the membranes of certain cancer cells effectively. However, electroporation makes it possible to insert bleomycin into cells.
Treatment typically is carried out by injecting an anticancer drug directly into the tumor and applying electroporation signals between a pair of electrodes positioned on opposite sides of a tumor. The field strength must be adjusted reasonably accurately so that electroporation of the cells of the tumor occurs without damage, or at least minimal damage, to any normal or healthy cells. This can normally be easily carried out with external tumors by applying the electrodes to opposite sides of the tumor so that the electric field is between the electrodes. When the field is uniform, the distance between the electrodes can then be measured and a suitable voltage according to the formula E=V/d can then be applied to the electrodes (E=electric field strength in V/cm; V=voltage in volts; and d=distance in cm). When large or internal tumors are to be treated, it is not easy to properly locate electrodes and measure the distance between them. The aforementioned parent application discloses a system of electrodes for in vivo electroporation wherein the electrodes may be inserted into the tumor. In related U.S. Pat. No. 5,273,525, a syringe for injecting molecules and macromolecules for electroporation utilizes needles for injection which also function as electrodes. This construction enables subsurface placement of electrodes.
DNA immunization, a novel method to induce protective immune responses, was recently introduced into the scientific community and proven to be very effective in animal models. This technology is currently in first safety and efficacy trials in human volunteers. DNA immunization entails the direct, in vivo administration of plasmid-based DNA vectors that encode the production of defined microbial antigens or other desired antigens. The de novo production of these antigens in the host""s own cells results in the elicitation of antibody (i.e. humoral) and cellular immune responses that provide protection against live virus challenge, for example, and persist for extended periods in the absence of further immunizations. The unique advantage of this technology is its ability to mimic the effects of live attenuated vaccines without the safety and stability concerns associated with the parenteral administration of live infectious agents. Because of these advantages, considerable research efforts have focused on refining in vivo delivery systems for naked DNA that result in maximal antigen production and resultant immune responses.
The most widely used administration of vaccine DNA is direct injection of the DNA into muscle or skin by needle and syringe. This method is effective in inducing or augmenting immune responses in small animals, as mice, but even here it requires the administration of relatively large amounts of DNA, ca. 50 to 100 ug per mouse. To obtain immune responses in larger animals, as rabbits, non-human primates, and humans, very large amounts of DNA have to be injected. It has to be seen whether this requirement for very large amounts of vaccine DNA turns out to be practical, for safety and commercial reasons, in human applications.
Despite the suitability of the epidermis as a target tissue for gene therapy or DNA vaccination, there are significant barriers to safe, easy, efficient, and economical gene delivery. In particular, the lipid-rich stratum corneum, which is composed of dead keratinocytes surrounded by multiple, parallel bilayer membranes, represents a formidable physical barrier to epidermal gene transfer. To overcome this barrier, a novel, non-viral approach, involving the basic concept of electroporation to introduce genes into the epidermis or muscle is provided by the present invention.
Treatment of a subject using electroporation provides a means for avoiding the deleterious effects typically associated with administration of anticancer or cytotoxic agents. Such treatment would allow introduction of these agents to selectively damage or kill undesirable cells while avoiding surrounding healthy cells or tissue. However, the electrical signals which are typically used for electroporation cause considerable discomfort to a patient. There is often enough discomfort that patients are given general anesthesia before receiving the electroporation treatment.
The present invention is based on the discovery of apparatuses, instruments and methods for reducing pain often associated with clinical use of electroporation and delivery of agents to cells. The invention provides such apparatus and instruments which allow administration of electric pulses to a patient while reducing the level of discomfort.
In one embodiment, a method of the invention includes positioning a first electrode and a second electrode such that an electrical signal passed between the first electrode and the second electrode passes through the cell. The method also includes passing an electrical signal with a frequency greater than about 10 kHz between the first electrode and the second electrode. In one embodiment of the method, the electrical signal has a bipolar square waveform. In another embodiment of the method, the electrodes are positioned at a treatment site for in vivo delivery of an agent.
The invention also relates to an electroporation instrument for use with an electroporation therapy apparatus having two or more electrodes. The electroporation instrument includes a connector configured to be coupled with the electroporation therapy apparatus. The connector provides electrical communication between the electroporation instrument and the electrodes of the electroporation therapy apparatus. The electroporation instrument also includes electronics for applying an electrical signal to the two or more electrodes. The electrical signal has a frequency greater than about 10 kHz. In one embodiment, the electrical signal has a bipolar square waveform.
One embodiment of the invention includes electronics for applying an electroporation signal to the electrodes of the electroporation therapy apparatus and electronics for applying an agent movement signal to the electrodes of the electroporation therapy apparatus. The agent movement signals can be applied independently of the electroporation signals.
Another embodiment of the electroporation instrument includes electronics for applying therapeutic electrical signals to the plurality of electrodes and electronics for testing whether the electrodes are positioned at the treatment site in an orientation suitable for applying the therapeutic electrical signals to the electrodes. The electroporation instrument can also include. electronics for withholding the therapeutic signals if the electrodes are not positioned in an orientation suitable for application of the therapeutic signals.
Yet another embodiment of the electroporation instrument includes electronics for applying an electrical signal having a bipolar waveform to the electrodes of the electroporation therapy apparatus. The electronics include a power source for producing a monopolar electrical signal and polarity changing electronics for changing the monopolar electrical signal to a bipolar electrical signal. Hence, the electroporation instrument can include a single power source for providing bipolar signals.